Our research is focused on elucidating the basic mechanisms by which selenoproteins are synthesized in vivo and to investigate the structure and function of selenoenzymes. Currently, we are investigating the following projects:(a) Selenophosphate is the selenium donor for the biosynthesis of selenocysteine-containing proteins and seleno-tRNA. Selenophosphate synthetase (SPS) catalyzes the formation of selenophosphate from ATP and selenide. The structural study of SPS has been hampered by the difficulty in obtaining suitable crystals for x-ray crystallographic analysis. We have now successfully crystallized a SPS (C17S) mutant in which selenomethionine is substituted for methionine. The structural analysis revealed that the SPS mutant exists as a homodimer with a flexible N-terminal region. A wild-type SPS crystal is also being prepared for structural studies. In vitro kinetic studies revealed a selenide Km of 7.3 microM for SPS, which is significantly higher than the toxic level for mammals. Therefore, it is reasonable to assume the existence of a selenium delivery protein, such that the SPS can function with a non-toxic level of selenide. To this end, we have investigated various potential candidates, among them a novel selenium-binding protein (SeBP) isolated from Methanococcus vannielii. To better understand how SeBP works as a selenium-binding protein, the solution structure of SeBP was determined by Nuclear Magnetic Resonance (NMR) methods. The results show that SEBP exists as a pentamer and each subunit is composed of a gamma-helix on top of a 4-stranded, twisted gamma-sheet. The pentameric structure is maintained mainly via hydrophobic interactions supplemented by hydrogen bond interactions. Surprisingly, cysteine 59, believed to be involved in selenium binding, is located in a flexible loop close to the core and not readily accessible to free selenium. In addition, site-directed mutagenesis, guided by the structure, revealed that Ile 9, Ser 22, and Ile 25 are essential for stabilizing the pentamer structure. (b) Overexpression of selenium-containing proteins is hampered by the requirement of a complex co-translational selenocysteine incorporation mechanism. To bypass this problem, we collaborated with Peter Schultz and Jiangyun Wang to use Schultzs methodology to incorporate an unnatural amino acid into a specific site of a given protein in both prokaryotic and eukaryotic organisms. This method relies on a unique codon-tRNA pair and corresponding aminoacyl tRNA synthetase for the unnatural amino acid that does not cross-react with any of the endogenous tRNAs, aminoacyl tRNA synthetases, amino acids, or codons in the host organism. In addition, we also are developing a selenoprotein expressing system using Dictyostelium and the autonomous replicable plasmid obtained from Dr. Ed Korn. (c) Investigating selenium metabolism in a single-celled eukaryotic organism, the amoeba form of Dictyostelium, we found that the mRNA levels, encoding SelD, SelK and an deiodinase-like protein, were induced by selenite treatment during cell growth, and selenite and hydrogen peroxide also induced mRNA levels encoding thioredoxin and glutaredoxin. Studies using the knockout mutant for SelK are in progress.